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737. Tumor-Selective TK Gene Therapy by Administration of Gancicrovir–Lipiodol with SeV-TK
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER 737. Tumor-Selective TK Gene Therapy by Administration of Gancicrovir–Lipiodol with SeVTK
738. HSV-TK/GCV Gene Therapy Approaches for Treating Brain Tumors Are Limited by the Efficacy of Gene Transfer
Tatsuya Okimoto,1 Hiroshi Yahata,1 Mitsuo Yoshimatsu,2 Kenji Kihira,2 Katsunori Shinozaki,1 Hidehiro Tanji,1 Toshimasa Asahara.1 1 Department of Surgery, Division of Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan; 2Department of Pharmacology, Division of Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.
Piotr Hadaczek,1 Hanna Mirek,1 Mitchel S. Berger,2 Krys Bankiewicz.1 1 Gene and Drug Delivery Laboratory - Neurological Surgery, University of California San Francisco, San Francisco, CA; 2 Neurological Surgery, University of California San Francisco, San Francisco, CA.
Background: Various gene therapy methods for treatment of liver cancer, such as transduction of the cytokine gene, wild-type p53 gene or TK suicide gene, have been extensively studied. TK gene therapy has been shown to be very effective for destroying the tumor by converting gancicrovir (GCV) to gancicrovir-triphosphate. The most undesirable adverse effect of this therapy is toxicity to the normal parenchyma of the liver that has been transduced with the TK gene. The effectiveness of various tumor selective promoters that express the gene of interest only in the tumor for prevention of this side effect has been studied. However, tumor selective promoters usually can not drive the gene as well as conventionally used promoters can. Purpose of the study: In the last ASGT meeting held in Boston, 2002, we presented an effective method for administering GCV in a tumor-selective manner by using lipiodol, which is a commonly used enhancing reagent in liver-tumor selective chemotherapy in Japan. This year, we improved this method by adding iopamidol and HCO60 to Lipiodol-GCV to make more GCV stay in the tumor. Further, Sendai virus vector that expresses TK gene, SeV-TK, was transduced to the tumor that hold GCV-lipiodol in a tumor selective manner to see the effect of Lipiodol-GCV-TK system. Materials and methods: At first, we injected 1x107/100ul of McA-RH7777 cells, a p53mut hepatocellular carcinoma cell line, into subcutaneous region of Buffalo rats. After a subcutaneous tumor formed, we resected the tumor and cut into pieces around 2mm in diameter. Then, we transplanted this piece of the tumor into the liver of another Buffalo rats. A solitary, solid liver tumor developed 4 to 8 weeks after transplantation. GCVlipiodol was locally injected into the tumor. The densities of GCV in the tumor and normal parenchyma were measured by HPLC. SeVTK was injected simultaneously with GCV-lipiodol. SeV-LacZ was used as a control. Result: 1. 24h after local injection of GCV-lipiodol, the densities of GCV in the tumor was 1.50±0.4ug/ml, while 0.06±0.12ug/ml in normal liver parenchyma. 2. The density of GCV in the tumor at 48 h after local injection of GCV-lipiodol was kept at the same level as in 24hr after injection. 3.Growth suppression and increasing of apoptotic cell in the tumor was observed in SeV-TK group. Conclusions: Tumor-selective TK gene therapy is possible by improved GCVlipiodol system with SeV-TK.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Purpose: We evaluated the efficacy of eradication of U87MG cell-derived brain tumors in a rat model using HSV-TK/GCV system. Local intratumoral convection enhanced delivery of AAV2-TK vectors was applied to enlarge the volume of the transduced tumor mass. Methods: U87MG cells were transduced in vitro with AAV2TK and transduction efficiency was examined at different time points after infection. Parallel animal model with xenograft tumor-bearing rats was used to compare TK expression dynamics within U87MG tumors in vivo. To examine the efficacy of HSV-TK gene therapy on these tumors we treated tumor-bearing rats with GCV. Cytovene was injected i.p. (50mg/kg) every day (10-day treatment). The mean survival time was calculated for the treated and control (PBS) group. Results: We found that expression of TK in U87MG cells transduced in vitro with AAV2-TK decreased rapidly over time. 24 hours after infection almost 100% of cells showed positive signal for TK . After 7 days only 16.5% showed to be TK-positive. After 15 and 30 days this number dropped down to 3.7% and ~1%, respectively. The results from in vivo experiments using rats implanted with previously transduced U87MG cells confirmed losing TK expression within tumor mass with similar rate. This phenomenon was most likely due to the lack of vector genomic integration and episomal form of the transgene was expelled from rapidly dividing tumor cells. Using CED and co-infusion with heparin we were able to enhance transduction efficiency in growing U87MG tumors by distributing AAV2-TK vectors over larger area. In terminal sized tumors (233 mm3; 22 days since implantation) transduced portion of a tumor mass accounted for 18% (42 mm3) while in early introduced AAV2 infusions (18 days since implantation; tumor volume 108 mm3) TK-positive area reached 39%. This resulted in, much higher than before, greater number of infected tumor cells subjected to following eradication with GCV treatment. In our animal survival study we showed that intraperinateal injections of GCV had given therapeutic effect and prolonged the survival time of the treated rats. In spite of statistically significant difference between GCV-treated and control animals (25.5 vs. 21.4 days, P<0.05) we concluded that even if robust tumor area was transduced with AAV2-TK vector, we were unable to infect all tumor cells. This eliminates possibility to completely eradicate growing tumors with HSV-TK/GCV system as unaffected tumor masses will eventually overgrow normal tissues. This, in turn, raises the question as to the usefulness of AAV-based vectors for gene therapy in human cancers. Unstable and transitional expression of episomal form of these in rapidly dividing cells seems to be a serious limiting factor for such application.
S285
738. HSV-TK/GCV Gene Therapy Approaches for Treating Brain Tumors Are Limited by the Efficacy of Gene Transfer
Tatsuya Okimoto,1 Hiroshi Yahata,1 Mitsuo Yoshimatsu,2 Kenji Kihira,2 Katsunori Shinozaki,1 Hidehiro Tanji,1 Toshimasa Asahara.1 1 Department of Surgery, Division of Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan; 2Department of Pharmacology, Division of Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan.
Piotr Hadaczek,1 Hanna Mirek,1 Mitchel S. Berger,2 Krys Bankiewicz.1 1 Gene and Drug Delivery Laboratory - Neurological Surgery, University of California San Francisco, San Francisco, CA; 2 Neurological Surgery, University of California San Francisco, San Francisco, CA.
Background: Various gene therapy methods for treatment of liver cancer, such as transduction of the cytokine gene, wild-type p53 gene or TK suicide gene, have been extensively studied. TK gene therapy has been shown to be very effective for destroying the tumor by converting gancicrovir (GCV) to gancicrovir-triphosphate. The most undesirable adverse effect of this therapy is toxicity to the normal parenchyma of the liver that has been transduced with the TK gene. The effectiveness of various tumor selective promoters that express the gene of interest only in the tumor for prevention of this side effect has been studied. However, tumor selective promoters usually can not drive the gene as well as conventionally used promoters can. Purpose of the study: In the last ASGT meeting held in Boston, 2002, we presented an effective method for administering GCV in a tumor-selective manner by using lipiodol, which is a commonly used enhancing reagent in liver-tumor selective chemotherapy in Japan. This year, we improved this method by adding iopamidol and HCO60 to Lipiodol-GCV to make more GCV stay in the tumor. Further, Sendai virus vector that expresses TK gene, SeV-TK, was transduced to the tumor that hold GCV-lipiodol in a tumor selective manner to see the effect of Lipiodol-GCV-TK system. Materials and methods: At first, we injected 1x107/100ul of McA-RH7777 cells, a p53mut hepatocellular carcinoma cell line, into subcutaneous region of Buffalo rats. After a subcutaneous tumor formed, we resected the tumor and cut into pieces around 2mm in diameter. Then, we transplanted this piece of the tumor into the liver of another Buffalo rats. A solitary, solid liver tumor developed 4 to 8 weeks after transplantation. GCVlipiodol was locally injected into the tumor. The densities of GCV in the tumor and normal parenchyma were measured by HPLC. SeVTK was injected simultaneously with GCV-lipiodol. SeV-LacZ was used as a control. Result: 1. 24h after local injection of GCV-lipiodol, the densities of GCV in the tumor was 1.50±0.4ug/ml, while 0.06±0.12ug/ml in normal liver parenchyma. 2. The density of GCV in the tumor at 48 h after local injection of GCV-lipiodol was kept at the same level as in 24hr after injection. 3.Growth suppression and increasing of apoptotic cell in the tumor was observed in SeV-TK group. Conclusions: Tumor-selective TK gene therapy is possible by improved GCVlipiodol system with SeV-TK.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
Purpose: We evaluated the efficacy of eradication of U87MG cell-derived brain tumors in a rat model using HSV-TK/GCV system. Local intratumoral convection enhanced delivery of AAV2-TK vectors was applied to enlarge the volume of the transduced tumor mass. Methods: U87MG cells were transduced in vitro with AAV2TK and transduction efficiency was examined at different time points after infection. Parallel animal model with xenograft tumor-bearing rats was used to compare TK expression dynamics within U87MG tumors in vivo. To examine the efficacy of HSV-TK gene therapy on these tumors we treated tumor-bearing rats with GCV. Cytovene was injected i.p. (50mg/kg) every day (10-day treatment). The mean survival time was calculated for the treated and control (PBS) group. Results: We found that expression of TK in U87MG cells transduced in vitro with AAV2-TK decreased rapidly over time. 24 hours after infection almost 100% of cells showed positive signal for TK . After 7 days only 16.5% showed to be TK-positive. After 15 and 30 days this number dropped down to 3.7% and ~1%, respectively. The results from in vivo experiments using rats implanted with previously transduced U87MG cells confirmed losing TK expression within tumor mass with similar rate. This phenomenon was most likely due to the lack of vector genomic integration and episomal form of the transgene was expelled from rapidly dividing tumor cells. Using CED and co-infusion with heparin we were able to enhance transduction efficiency in growing U87MG tumors by distributing AAV2-TK vectors over larger area. In terminal sized tumors (233 mm3; 22 days since implantation) transduced portion of a tumor mass accounted for 18% (42 mm3) while in early introduced AAV2 infusions (18 days since implantation; tumor volume 108 mm3) TK-positive area reached 39%. This resulted in, much higher than before, greater number of infected tumor cells subjected to following eradication with GCV treatment. In our animal survival study we showed that intraperinateal injections of GCV had given therapeutic effect and prolonged the survival time of the treated rats. In spite of statistically significant difference between GCV-treated and control animals (25.5 vs. 21.4 days, P<0.05) we concluded that even if robust tumor area was transduced with AAV2-TK vector, we were unable to infect all tumor cells. This eliminates possibility to completely eradicate growing tumors with HSV-TK/GCV system as unaffected tumor masses will eventually overgrow normal tissues. This, in turn, raises the question as to the usefulness of AAV-based vectors for gene therapy in human cancers. Unstable and transitional expression of episomal form of these in rapidly dividing cells seems to be a serious limiting factor for such application.
S285